EP2449135B1 - Rapid screening of biologically active nucleases and isolation of nuclease-modified cells - Google Patents

Rapid screening of biologically active nucleases and isolation of nuclease-modified cells Download PDF

Info

Publication number
EP2449135B1
EP2449135B1 EP10794489.4A EP10794489A EP2449135B1 EP 2449135 B1 EP2449135 B1 EP 2449135B1 EP 10794489 A EP10794489 A EP 10794489A EP 2449135 B1 EP2449135 B1 EP 2449135B1
Authority
EP
European Patent Office
Prior art keywords
cells
reporter
zinc finger
nuclease
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP10794489.4A
Other languages
German (de)
English (en)
French (fr)
Other versions
EP2449135A1 (en
EP2449135A4 (en
Inventor
Michael C. Holmes
Tianjian Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sangamo Therapeutics Inc
Original Assignee
Sangamo Biosciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sangamo Biosciences Inc filed Critical Sangamo Biosciences Inc
Publication of EP2449135A1 publication Critical patent/EP2449135A1/en
Publication of EP2449135A4 publication Critical patent/EP2449135A4/en
Application granted granted Critical
Publication of EP2449135B1 publication Critical patent/EP2449135B1/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the present disclosure is in the fields of genome engineering and nuclease identification.
  • Nucleases including zinc finger nucleases and homing endonucleases such as I-SceI, that are engineered to specifically bind to target sites have been shown to be useful in genome engineering in basic research and in the pharmaceutical and biotechnology applications.
  • zinc finger nucleases are proteins comprising engineered site-specific zinc fingers fused to a nuclease domain.
  • ZFNs have been successfully used for genome modification in a variety of different species. See, for example, United States Patent Publications 20030232410 ; 20050208489 ; 20050026157 ; 20050064474 ; 20060188987 ; 20060063231 ; and International Publication WO 07/014275 .
  • ZFNs can be used to create a double-strand break (DSB) in a target nucleotide sequence, which increases the frequency of homologous recombination at the targeted locus (targeted integration) more than 1000-fold.
  • DSB double-strand break
  • NHEJ nonhomologous end joining
  • DSBs DNA double-strand breaks
  • HDR conservative homology directed repair
  • SSA single-strand-annealing
  • US 2009/0111119 discloses reporter constructs for the in vivo identification of biologically active nucleases.
  • the assays make use of a zinc finger nuclease that cuts at a site (e.g ., an engineered site) in a disabled gene, preferably a reporter gene.
  • the disabled gene is preferably episomal ( i.e ., located within a construct that is not within the endogenous locus). Cleavage by the zinc finger nuclease at the engineered site allows the homologous regions to repair and reconstitute the disabled gene via SSA.
  • the engineered site within the disabled gene has the same sequence as a target site within an endogenous target locus where cleavage is desired, such that cleavage at the endogenous site occurs when the disabled reporter gene of the construct is cleaved.
  • the relative efficiency of SSA repair correlates well with relative efficiency of zinc finger nuclease activity at the endogenous target locus.
  • individual cells that carry out SSA-mediated repair in assays as described herein show increased modification at the endogenous target locus thus, allowing for the rapid identification of cells with the desired genomic modification(s).
  • the methods and compositions described herein significantly alleviate the obstacles associated with integration of selection or other markers into the genome.
  • the invention relates to a host cell comprising an episomal reporter construct, wherein the episomal reporter construct comprises multiple target sequences for one or more zinc finger nucleases flanked by sequences encoding a reporter gene, wherein the target sequences are inserted between two identical partial sequences of the reporter gene, the two identical partial sequences of the reporter gene flanked by unique 3' and 5' coding regions of the reporter gene and further, wherein said target sequences have the same sequence as target sites present in the genome of the host cell.
  • the reporter construct for detecting SSA mediated cleavage of a target sequence by one or more nucleases.
  • the reporter construct comprises a sequence encoding a gene and a sequence comprising multiple target sites for a zinc finger nuclease inserted within the sequence encoding the gene such that the gene is non-functional (disabled) until the target site(s) is (are) cleaved and repaired by SSA. Following cleavage of the target site(s), the sequence encoding the gene is recreated by SSA and gene function restored.
  • the reporter construct comprises, in a 5' to 3' direction, a first nucleotide sequence encoding a first portion of a reporter gene, a second nucleotide sequence encoding a second portion of the reporter gene, a sequence comprising multiple target sequences for a zinc finger nuclease, a third nucleotide sequence encoding the second portion of the reporter gene and a fourth nucleotide sequence encoding a third portion of the reporter gene.
  • the first, second and third portions of the reporter gene encode the functional reporter gene.
  • Any of the reporter constructs described herein may further comprise a polyadenylation signal and/or a promoter ( e.g ., a constitutive promoter) operably linked the reporter gene.
  • the reporter gene can encode a light-generating protein (e . g . GFP), an enzyme, a cell surface receptor, and/or a selectable marker.
  • the host cell is a eukaryotic cell (e.g., a mammalian cell).
  • the reporter construct may be transiently expressed in the host cell.
  • Any of the host cells may further comprise a sequence encoding a zinc finger nuclease.
  • a method of identifying one or more zinc finger nucleases that induce cleavage at a specific target site comprising the steps of: introducing one or more expression constructs that express the zinc finger nuclease(s) into any of the host cells described herein, wherein the reporter construct comprises a target sequence recognized by the zinc finger nuclease; incubating the cells under conditions such that the zinc finger nuclease is expressed; and measuring the levels of reporter gene expression in the cells, wherein increased levels of reporter gene expression are correlated with increased zinc finger nuclease-induced cleavage of the target sequence.
  • the methods comprise introducing one or more zinc finger nucleases and/or one or more zinc finger nuclease-expression constructs encoding a zinc finger nuclease or a pair of zinc finger nucleases into a host cell comprising a reporter construct as described herein, the reporter construct comprising a target sequence recognized by the zinc finger nuclease(s); incubating the cells under conditions such that the zinc finger nuclease(s) are expressed, and measuring the levels of reporter gene expression in the cells, wherein increased levels of reporter gene expression are correlated with increased zinc finger nuclease-induced cleavage of the target sequence.
  • the zinc finger nuclease may comprise, for example, an engineered zinc finger protein
  • the invention includes a method of enriching a population of cells for cells having a zinc finger nuclease-mediated genomic modification, the method comprising the steps of: introducing one or more expression constructs encoding a zinc finger nuclease or a pair of zinc finger nucleases into host cells as described herein, wherein the reporter construct in the host cells comprises a target sequence recognized by the zinc finger nuclease; incubating the cells under conditions such that the zinc finger nucleases are expressed; measuring the levels of reporter gene expression in the cells; and selecting cells that express the reporter gene, thereby enriching the population of cells for cells with zinc finger nuclease-mediated genomic modifications.
  • a panel of zinc finger nucleases may be compared simultaneously that all recognize the same target sequence.
  • the panel may be transfected along with the SSA reporter in parallel, providing a rapid indication and ranking of activity of those zinc finger nucleases within the test panel.
  • Any of the methods may further comprise introducing an exogenous sequence into the host cell such that the zinc finger nuclease mediates targeted integration of the exogenous sequence into the genome.
  • the methods further comprise isolating the cells expressing the reporter gene.
  • the genomic modification is a gene disruption and/or a gene addition.
  • reporter gene activity may be measured directly, for example by directly assaying the levels of the reporter gene product activity (e.g ., GFP fluorescence).
  • cells expressing the reporter gene may be isolated or selected based on direct selection, for example FACS in the case of a reporter such as GFP or using fluorescent ligands directed to a reporter gene encoding a cell surface protein or receptor. Magnetic sorting can also be employed.
  • the reporter is a drug selection marker
  • drug selection may also be used to select cells.
  • levels of the reporter gene can be assayed by measuring or selecting based on the levels of a downstream product (e . g ., enzymatic product) of the reaction that requires function of the protein encoded by the reporter gene.
  • the zinc finger nuclease(s) may be known to recognize the endogenous target sequence, for example from results obtained from in vitro assay experiments.
  • a kit for screening a zinc finger nuclease for activity comprising a reporter construct as described herein; ancillary reagents; and optionally instructions and suitable containers.
  • the kit may also include one or more zinc finger nucleases,
  • kits for preparing cells having zinc finger nuclease-mediated genomic modifications comprising a reporter construct as described herein and a zinc finger nuclease that recognizes a target site in the reporter construct; and optionally instructions and suitable containers.
  • kits described herein may comprise at least the construct with the disabled gene and a known zinc finger nuclease capable of cleaving within the disabled gene at a known engineered site. Such kits are useful for optimization of cleavage conditions. Other such kits may provide constructs wherein the user may insert the engineered site of interest for use in identifying and /or screening nucleases capable of cleavage at such an engineered site.
  • the disabled gene is a screening marker (e.g. GFP), while in other embodiments, the disable gene is a selection marker such as one encoding antibiotic resistance.
  • the disabled gene encodes a cell surface marker or receptor wherein following reconstitution via SSA, the reporter is expressed on the cell surface and can be used to identify those clones wherein SSA mediated gene reconstitution has occurred (e.g., via FACS or magnetic bead sorting).
  • the reporter gene may be operatively linked to a polyadenylation signal and/or a regulatory element (e.g. a promoter).
  • a regulatory element e.g. a promoter
  • compositions and methods for high throughput in vivo screening systems for identifying functional zinc finger nucleases and kits comprising the compositions described herein and for carrying out the methods described herein.
  • the assays use a reporter system to monitor the ability of a zinc finger nuclease to induce a double-stranded break at a target site.
  • the compositions and methods described herein can also be used to screen panels of nucleases to identify those with the highest activity, to optimize zinc finger nuclease cleavage conditions and to rapidly enrich for modified cell lines or clones that have undergone zinc finger nuclease-induced gene disruption and/or gene addition.
  • Engineered nuclease technology is based on the engineering of naturally occurring DNA-binding proteins.Engineering of ZFPs has been described. See, e.g., U.S. Patent Nos. 6,534,261 ; 6,607,882 ; 6,824,978 ; 6,979,539 ; 6,933,113 ; to 63,824 ; and 7,013,219 .
  • ZFPs have been attached to nuclease domains to create ZFNs - a functional entity that is able to recognize its intended gene target through its engineered (ZFP) DNA binding domain and the nuclease causes the gene to be cut near the ZFP binding site.
  • ZFP engineered
  • ZFNs have been used for genome modification in a variety of organisms. See, for example, United States Patent Publications 20030232410 ; 20050208489 ; 20050026157 ; 20050064474 ; 20060188987 ; 20060063231 ; and International Publication WO 07/014275
  • the methods described herein provide a rapid and efficient way of evaluating zinc finger nucleases known to bind to a particular target site for their in vivo functionality as well as the ability to rapidly identify and isolate cells with the desired nuclease-mediated genomic modifications.
  • the methods and compositions described herein provide highly efficient and rapid methods for identifying zinc finger nucleases that are biologically active in vivo.
  • the assays described herein also can be used to screen for and isolate zinc finger nuclease-modified cells that do not contain an integrated reporter construct. These methods and compositions also allow the ranking of the most active zinc finger nucleases in cells simply through the measurement of a reconstituted reporter gene's activity.
  • the methods and compositions described herein also provide the components for kits to allow screening, optimization and characterization of zinc finger nucleases within a cell.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001 ; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego ,; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998 ; METHODS IN ENZYMOLOGY, Vol. 304, "Chromatin" (P.M. Wassarman and A. P.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
  • polynucleotide refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
  • these terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones).
  • an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally-occurring amino acids.
  • Binding refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g ., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a dissociation constant (K d ) of 10 -6- M -1 or lower.
  • K d dissociation constant
  • Affinity refers to the strength of blinding
  • a "binding protein” is a protein that is able to bind non-covalently to another molecule.
  • a binding protein can bind to, for example, a DNA molecule (a DNA- binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein).
  • a DNA-binding protein binds to DNA molecule
  • RNA-binding protein an RNA-binding protein
  • a protein-binding protein it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins.
  • a binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein- binding activity.
  • a “zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
  • the term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
  • Zinc finger binding domains can be "engineered” to bind to a predetermined nucleotide sequence.
  • the engineered region of the zinc finger is typically the recognition helix, particularly the portion of the alpha- helical region numbered -1 to +6.
  • Backbone sequences for an engineered recognition helix are known in the art. See, e.g ., Miller et al. (2007) Nat Biotechnol 25, 778-785 .
  • Non-limiting examples of methods for engineering zinc finger proteins are design and selection.
  • a designed zinc finger protein is a protein not occurring in nature whose design/composition results principally from rational criteria.
  • Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, US Patents 6,140,081 ; 6,453,242 ; and 6,534,261 ; see also WO 98/53058 ; WO 98/53059 ; WO 98/53060 ; WO 02/016536 and WO 03/016496 .
  • a "selected" zinc finger protein is a protein not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., US 5,789,538 ; US 5,925,523 ; US 6,007,988 ; US 6,013,453 ; US 6,200,759 ; WO 95/19431 ; WO 96/06166 ; WO 98/53057 ; WO 98/54311 ; WO 00/27878 ; WO 01/60970 WO 01/88197 and WO 02/099084 .
  • a "TAL- effector repeat sequence” is the structural sequence that is involved in the binding of the TAL- effector to its cognate target DNA sequence. These repeats are typically 34 amino acids in length and almost invariably exhibit a great deal of sequence homology with other TAL- effector repeat sequences within a TAL- effector protein. Positions 12 and 13 exhibit hypervariability and are thought to be the amino acids that determine what DNA nucleolide the repeat will interact with. The identity of these amino acids largely determine the DNA base the repeat sequence interacts with. The most C-terminal repeat often displays sequence similarity only for the first 20 amino acids and so is sometimes referred to as a half repeat. The most N-terminal repeat has a sequence immediately preceding it that shows similarity to the repeat sequences on a structural level, and thus is termed the R0 repeat.
  • a "TAL-effector DNA binding domain” is a protein, or a domain within a larger protein, that interacts with DNA in a sequence-specific manner through one or more tandem repeat domains.
  • Cleavage refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage.
  • a “cleavage half-domain” is a polypeptide sequence which, in conjunction with a second polypeptide (either identical or different) forms a complex having cleavage activity (preferably double-strand cleavage activity).
  • first and second cleavage half-domains;" “+ and - cleavage half-domains” and “right and left cleavage half-domains” are used interchangeably to refer to pairs of cleavage half- domains that dimerize.
  • An "engineered cleavage half-domain” is a cleavage half-domain that has been modified so as to form obligate heterodimers with another cleavage half- domain (e.g., another engineered cleavage half-domain). See, also, U.S. Patent Publication Nos. 2005/0064474 , 20070218528 and 2008/0131962 .
  • Zinc finger DNA binding domains or TAL-effector DNA binding domains can be "engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the hypervariable diresidue region at positions 12 and 13 of a naturally repeat domain within a TAL- effector protein or by engineering the DNA binding portion of the DNA recognition helix of a zinc finger protein. Therefore, engineered zinc finger proteins and TAL- effector proteins are proteins that are non-naturally occurring.
  • Non-limiting examples of methods for engineering zinc finger proteins and TAL-effector proteins are design and selection.
  • a designed zinc finger protein or TAL-effector protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing zinc finger protein or TAL-effector designs and binding data.
  • Chromatin is the nucleoprotein structure comprising the cellular genome.
  • Cellular chromatin comprises nucleic acid, primarily DNA, and protein, including histones and non-histone chromosomal proteins.
  • the majority of eukaryotic cellular chromatin exists in the form of nucleosomes, wherein a nucleosome core comprises approximately 150 base pairs of DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4; and linker DNA (of variable length depending on the organism) extends between nucleosome cores.
  • a molecule of histone H1 is generally associated with the linker DNA.
  • chromatin is meant to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic.
  • Cellular chromatin includes both chromosomal and episomal chromatin.
  • a "chromosome,” is a chromatin complex comprising all or a portion of the genome of a cell.
  • the genome of a cell is often characterized by its karyotype, which is the collection of all the chromosomes that comprise the genome of the cell.
  • the genome of a cell can comprise one or more chromosomes.
  • an “episome” is a replicating nucleic acid, nucleoprotein complex or other structure comprising a nucleic acid that is not part of the chromosomal karyotype of a cell.
  • Examples of episomes include plasmids and certain viral genomes.
  • a “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.
  • the sequence 5'-GAATTC-3' is a target site for the Eco RI restriction endonuclease.
  • exogenous molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. "Normal presence in the cell" is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-heat-shocked cell.
  • An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally- functioning endogenous molecule.
  • An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
  • Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Patent Nos. 5,176,996 and 5,422,251 .
  • Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.
  • exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid.
  • an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell.
  • Methods for the introduction of exogenous molecules into cells include, but are not limited to, lipid-mediated transfer (i.e ., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate coprecipitation, DEAE-dextran- mediated transfer and viral vector-mediated transfer.
  • an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions.
  • an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally- occurring episomal nucleic acid.
  • Additional endogenous molecules can include proteins, for example, transcription factors and enzymes.
  • a "fusion" molecule is a molecule in which two or more subunit molecules are linked, preferably covalently.
  • the subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules.
  • Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (for example, a fusion between a ZFP DNA-binding domain and a cleavage domain or a fusion between a TAL-effector DNA binding domain and a cleavage domain) and fusion nucleic acids (for example, a nucleic acid encoding the fusion protein described supra).
  • Examples of the second type of fusion molecule include, but are not limited to, a fusion between a triplex-forming nucleic acid and a polypeptide, and a fusion between a minor groove binder and a nucleic acid.
  • Fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein.
  • Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.
  • Eukaryotic cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells (e.g., T-cells).
  • operative linkage and "operatively linked” (or “operably linked”) are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components.
  • a transcriptional regulatory sequence such as a promoter
  • a transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it.
  • an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous.
  • the term "operatively linked" can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked.
  • the ZFP DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the ZFP DNA-binding domain portion is able to bind its target site and/or its binding site, while the cleavage domain is able to cleave DNA in the vicinity of the target site.
  • a “vector” is capable of transferring gene sequences to target cells.
  • vector construct means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells.
  • vector transfer vector means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells.
  • the term includes cloning, and expression vehicles, as well as integrating vectors.
  • reporter gene refers to any sequence that produces a protein product that is easily measured, preferably although not necessarily in a routine assay.
  • Suitable reporter genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e . g ., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g ., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins which mediate enhanced cell growth and/or gene amplification (e.g., dihydrofolate reductase).
  • Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA or any detectable amino acid sequence.
  • compositions and methods for the in vivo identification of zinc finger nucleases that cleave their target sites with the highest frequency.
  • the compositions and methods described herein can also be used to isolate cells having the desired genomic modifications, but without an integrated reporter.
  • the reporter construct comprising the target site(s) for the zinc finger nuclease(s) is introduced into a host cell.
  • the zinc finger nuclease(s) are expressed in the cell and induce a double stranded break (DSB) at their target site (e . g ., induce a double-stranded break)
  • the reporter gene is reconstituted by the host cell's single- stranded annealing (SSA) machinery.
  • SSA single- stranded annealing
  • the reporter gene is readily determined by standard techniques and the levels of reporter gene expression reflect the ability of the zinc finger nuclease to cleave at the target site.
  • the SSA reporter systems accurately assess ZFN domain nuclease fusion protein activity on the endogenous target site and, accordingly, sorting cells for nuclease-mediated expression of the SSA reporter allows for high throughput screening and isolation of nuclease ( e . g ., ZFN, nuclease fusion protein)-modified cells.
  • compositions and methods described herein can also be utilized in kits that allow the user to screen zinc finger nucleases and to select cells with desired genomic modifications.
  • the methods and systems described herein make use of a reporter construct comprising a sequence containing multiple target sequences for the nuclease(s) to be tested.
  • the reporter construct is designed so that the reporter gene becomes functional only when the zinc finger nuclease cleaves the target sequence and the reporter gene is reconstituted by single-strand annealing (SSA) repair mechanisms.
  • SSA single-strand annealing
  • a reporter construct is generated such that any zinc finger nuclease target sequence(s) can be readily inserted into the middle of the disabled reporter gene sequence (see, Fig. 1A ),
  • the target sequences are inserted between two identical partial sequences of the reporter gene.
  • the two identical partial sequences on either site of the nuclease target site are flanked by unique 3' and 5' coding regions of the reporter gene.
  • the sequences between the two identical partial sequences are lost and the reporter gene reconstituted in a functional open reading frame. See, Fig. 1A .
  • One or more target sites for the nuclease(s) to be screened can be inserted into the reporter constructs by any suitable methodology, including PCR or commercially available cloning systems such as TOPO® and/or Gateway® cloning systems.
  • the target site comprises a concatamer of target sites.
  • Target sites can be from prokaryotic or eukaryotic genes, for example, mammalian (e.g., human), yeast or plant cells. It is required, that the target site(s) in the reporter constructs be present in the genome of the host cell.
  • reporter gene can be used in the SSA constructs described herein.
  • the reporter gene provides a directly detectable signal directly, for example, a signal from a fluorescent protein such as, for example, GFP (green fluorescent protein). Fluorescence is detected using a variety of commercially available fluorescent detection systems, including, e.g ., a fluorescence-activated cell sorter (FACS) system. Reporter genes may also be enzymes that catalyze the production of a detectable product (e.g. proteases, nucleases, lipases, phosphatases, sugar hydrolases and esterases).
  • FACS fluorescence-activated cell sorter
  • Non-limiting examples of suitable reporter genes that encode enzymes include, for example, MELI, CAT (chloramphenicol acetyl transferase; Alton and Vapnek (1979) Nature 282:864 869 ), luciferase, ⁇ - galactosidase, ⁇ -glucuronidase, ⁇ -lactamase, horseradish peroxidase and alkaline phosphatase (e.g., Toh, et al. (1980) Eur. J. Biochem. 182:231 238 ; and Hall et al. (1983) J. Mol. Appl. Gen. 2:101 ).
  • Additional reporter genes include cell-surface based markers (e.g., receptors) that can be enriched for by either FACS or antibody-coated magnetic beads as well as drug-based selection markers (e.g., antibiotic resistance such as ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance).
  • Cell-surface based markers e.g., receptors
  • drug-based selection markers e.g., antibiotic resistance such as ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance.
  • Magnetic beads carrying ligands for cell surface receptors or carrying compounds capable of interacting with cell surface receptors can be used with the methods of the invention.
  • commercially available nickel charged magnetic beads can be used to enrich cells in which a reconstituted cell surface protein contains a His tag.
  • commercially available magnetic cyanogen bromide beads can be activated to bind to a ligand of choice and then used in the methods described herein to enrich or purify cells containing a reconstituted SSA cell surface reporter protein.
  • the reporter construct typically includes a promoter that drives expression of the reporter gene upon cleavage by the nuclease and subsequent SSA- mediated repair of a functional reporter.
  • Any suitable promoter can be used, preferably a promoter that functional in the host cell.
  • the promoter is a constitutive promoter such as CMV, although in certain cases inducible promoters may be employed.
  • a polyadenylation signal may also be included in the reporter construct (see, e.g., Fig. 1A ).
  • the cell types can be cell lines or natural (e.g., isolated) cells such as, for example, primary cells.
  • Cell lines are available, for example from the American Type Culture Collection (ATCC), or can be generated by methods known in the art, as described for example in Freshney et al., Culture of Animal Cells, A Manual of Basic Technique, 3rd ed., 1994 , and references cited therein.
  • ATCC American Type Culture Collection
  • cells can be isolated by methods known in the art.
  • cell types include cells that have or are subject to pathologies, such as cancerous cells and transformed cells, pathogenically infected cells, stem cells, fully differentiated cells, partially differentiated cells, immortalized cells and the like.
  • Prokaryotic e.g., bacterial
  • eukaryotic e.g., yeast, plant, fungal, piscine and mammalian cells such as feline, canine, murine, bovine, porcine and human cells can be used, with eukaryotic cells being preferred.
  • Suitable mammalian cell lines include K562 cells, CHO (Chinese hamster ovary) cells, HEP-G2 cells, BaF-3 cells, Schneider cells, COS cells (monkey kidney cells expressing SV40 T-antigen), CV-1 cells, HuTu80 cells, NTERA2 cells, NB4 cells, HL-60 cells and HeLa cells, 293 cells (see, e.g., Graham et al. (1977) J. Gen. Virol. 36:59 ), and myeloma cells like SP2 or NSO (see, e.g. , Galfre and Milstein (1981) Meth. Enzymol. 73(B):3 46 ).
  • PBMCs Peripheral blood mononucleocytes
  • T-cells can also serve as hosts.
  • Other eukaryotic cells include, for example, insect (e.g., sp. frugiperda ), fungal cells, including yeast (e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis, H, polymorpha ), and plant cells ( Fleer, R. (1992) Current Opinion in Biotechnology 3:486 496 ).
  • the methods and compositions described herein are broadly applicable and may involve any zinc finger nuclease.
  • the zinc finger nuclease may comprise heterologous DNA-binding and cleavage domains.
  • ZFNs comprise a zinc finger protein that has been engineered to bind to a target site in a gene of choice and cleavage domain or a cleavage half- domain.
  • Zinc finger binding domains can be engineered to bind to a sequence of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141 ; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340 ; Isalan et al. (2001) Nature Biotechnol. 19:656-660 ; Segal et al. (2001) Curr Opin. Biotechnol. 12:632-637 ; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416 .
  • An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein.
  • Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, co-owned U.S. Patents 6,453,242 and 6,534,261 .
  • Exemplary selection methods including phage display and two-hybrid systems, are disclosed in US Patents 5,789,538 ; 5,925,523 ; 6,007,988 ; 6,013,453 ; 6,410,248 ; 6,140,466 ; 6,200,759 ; and 6,242,568 ; as well as WO 98/37186 ; WO 98/53057 ; WO 00/27878 ; WO 01/88197 and GB 2,338,237 .
  • enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned WO 02/077227 .
  • zinc finger domains and/or multi-fingered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, e.g., U.S. Patent Nos. 6,479,626 ; 6,903,185 , and 7,153,949 for exemplary linker sequences 6 or more amino acids in length.
  • the proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.
  • Zinc finger nucleases also comprise a nuclease (cleavage domain, cleavage half-domain).
  • the cleavage domain may be heterologous to the DNA-binding domain, for example a zinc finger DNA-binding domain and a cleavage domain from a nuclease.
  • Heterologous cleavage domains can be obtained from any endonuclease or exonuclease.
  • Exemplary endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, MA; and Belfort et al.
  • a cleavage half-domain can be derived from any nuclease or portion thereof, as set forth above, that requires dimerization for cleavage activity.
  • two fusion proteins are required for cleavage if the fusion proteins comprise cleavage half-domains.
  • a single protein comprising two cleavage half- domains can be used, The two cleavage half-domains can be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragments thereof).
  • the target sites for the two fusion proteins are preferably disposed, with respect to each other, such that binding of the two fusion proteins to their respective target sites places the cleavage half-domains in a spatial orientation to each other that allows the cleavage half-domains to form a functional cleavage domain, e.g., by dimerizing.
  • the near edges of the target sites are separated by 5-8 nucleotides or by 15-18 nucleotides.
  • any integral number of nucleotides or nucleotide pairs can intervene between two target sites ( e . g ., from 2 to 50 nucleotide pairs or more), In general, the site of cleavage lies between the target sites.
  • Restriction endonucleases are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding.
  • Certain restriction enzymes e.g., Type IIS
  • Fok I catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, US Patents 5,356,802 ; 5,436,150 and 5,487,994 ; as well as Li et al.
  • fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type HS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.
  • Fok I An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is Fok I.
  • This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575 . Accordingly, for the purposes of the present disclosure, the portion of the Fok I enzyme used in the disclosed fusion proteins is considered a cleavage half-domain.
  • two fusion proteins each comprising a Fok I cleavage half-domain, can be used to reconstitute a catalytically active cleavage domain.
  • a single polypeptide molecule containing a zinc finger binding domain and two Fok I cleavage half-domains can also be used. Parameters for targeted cleavage and targeted sequence alteration using zinc finger-Fok I fusions are provided elsewhere in this disclosure.
  • a cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity, or that retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.
  • Type IIS restriction enzymes are described in International Publication WO 07/014275 . Additional restriction enzymes also contain separable binding and cleavage domains, and these are contemplated by the present disclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420 .
  • the cleavage domain comprises one or more engineered cleavage half-domain (also referred to as dimerization domain mutants) that minimize or prevent homodimerization, as described, for example, in U.S. Patent Publication Nos. 20050064474 and 20060188987 .
  • Amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of Fok I are all targets for influencing dimerization of the Fok I cleavage half-domains.
  • Exemplary engineered cleavage half-domains of Fok I that form obligate heterodimers include a pair in which a first cleavage half-domain includes mutations at amino acid residues at positions 490 and 538 of Fok I and a second cleavage half-domain includes mutations at amino acid residues 486 and 499.
  • a mutation at 490 replaces Glu (E) with Lys (K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at 486 replaced Gln (Q) with Glu (E); and the mutation at position 499 replaces Iso (I) with Lys (K),
  • the engineered cleavage half-domains described herein were prepared by mutating positions 490 (E ⁇ K) and 538 (I ⁇ K) in one cleavage half-domain to produce an engineered cleavage half-domain designated "E490K:I538K” and by mutating positions 486 (Q ⁇ E) and 499 (I ⁇ L) in another cleavage half-domain to produce an engineered cleavage half-domain designated "Q486E:I499L".
  • the engineered cleavage half-domains described herein are obligate heterodimer mutants in which aberrant cleavage is minimized or abolished See, e.g.,
  • the engineered cleavage half-domains described herein can be obligate heterodimer mutants in which aberrant cleavage is minimized or abolished. See, e.g., Example 1 of WO 07/139898 .
  • the engineered cleavage half-domain comprises mutations at positions 486, 499 and 496 (numbered relative to wild-type Fokl), for instance mutations that replace the wild type Gln (Q) residue at position 486 with a Glu (E) residue, the wild type Iso (I) residue at position 499 with a Leu (L) residue and the wild-type Asn (N) residue at position 496 with an Asp (D) or Glu (E) residue (also referred to as a "ELD” and "ELE" domains, respectively).
  • the engineered cleavage half-domain comprises mutations at positions 490, 538 and 537 (numbered relative to wild-type Fok I), for instance mutations that replace the wild type Glu (E) residue at position 490 with a Lys (K) residue, the wild type Iso (I) residue at position 538 with a Lys (K) residue, and the wild-type His (H) residue at position 537 with a Lys (K) residue or a Arg (R) residue (also referred to as "KKK” and "KKR” domains, respectively).
  • the engineered cleavage half-domain comprises mutations at positions 490 and 537 (numbered relative to wild-type Fok I), for instance mutations that replace the wild type Glu (E) residue at position 490 with a Lys (K) residue and the wild-type His (H) residue at position 537 with a Lys (K) residue or a Arg (R) residue (also referred to as "KIK” and "KIR” domains, respectively).
  • Engineered cleavage half-domains described herein can be prepared using any suitable method, for example, by site-directed mutagenesis of wild-type cleavage half-domains ( Fok I) as described in U.S. Patent Publication Nos. 20050064474 and 20080131962 .
  • nucleases may be assembled in vivo at the nucleic acid target site using so-called "split-enzyme” technology (see e.g. U.S. Patent Publication No. 20090068164 ).
  • split-enzyme e.g. U.S. Patent Publication No. 20090068164 .
  • Components of such split enzymes may be expressed either on separate expression constructs, or can be linked in one open reading frame where the individual components are separated, for example, by a self-cleaving 2A peptide or IRES sequence.
  • Components may be individual zinc finger binding domains.
  • Zinc finger nucleases can be screened for activity prior to use, for example in a yeast-based chromosomal system as described in WO 2009/042163 .
  • Nuclease expression constructs can be readily designed using methods known in the art. See, e.g., United States Patent Publications 20030232410 ; 20050208489 ; 20050026157 ; 20050064474 ; 20060188987 ; 20060063231 ; and International Publication WO 07/014275 , Expression of the nuclease may be under the control of a constitutive promoter or an inducible promoter, for example the galactokinase promoter which is activated (de-repressed) in the presence of raffinose and/or galactose and repressed in presence of glucose. Kits
  • kits for performing any of the above methods typically contain one or more reporter constructs as described herein, each reporter containing a cloning site for insertion of the target site for a zinc finger nuclease of interest.
  • kits for screening zinc finger nucleases with activity to a particular gene are provided with one or more reporter constructs containing the desired target site(s).
  • kits for enriching cells for a population of cells having a nuclease-mediated genomic modification comprise a reporter construct comprising a target site present in the genome of the cells and one or more zinc finger nuclease specific to the target site of interest.
  • kits can also contain cells, buffers for transformation of cells, culture media for cells, and/or buffers for performing assays.
  • the kits also contain a label which includes any material such as instructions, packaging or advertising leaflet that is attached to or otherwise accompanies the other components of the kit.
  • the disclosed methods and compositions can be used for rapid identification of zinc finger nucleases that are active on their endogenous targets without integration of the reporter construct into the genome of any host cell. Identification of such nucleases begins with the generation of an episomal reporter construct which is configured with the zinc finger nuclease binding site(s) inserted between 2 stretches of homologous reporter sequences. Cleavage by the nuclease allows the 2 homologous sequences to repair and reconstitute a functional reporter via SSA. The relative efficiency of this repair that allows the expression of the reporter correlates well with relative efficiency of nuclease activity at the endogenous target locus. Thus, the methods and compositions described herein allow for high-throughput screening of active zinc finger nucleases.
  • compositions and methods described herein allow for efficient isolation of cells containing nuclease-modified genomes
  • Cells that carry out SSA-mediated repair of the episomal plasmid-based or viral-based (e.g. adenoviral, AAV or lentiviral derived) reporter also show an increased level of modification at the endogenous target locus, including both NHEJ activities and as well as targeted integration of donor sequences.
  • modified cell clones can be efficiently isolated following enrichment using the reconstituted SSA marker by selecting cells expressing the reporter gene.
  • fluorescence activated cell sorting FACS
  • FACS fluorescence activated cell sorting
  • cells with a reconstituted SSA marker may be enriched or purified using a drug selection scheme wherein the reconstituted SSA marker encodes a resistance marker.
  • Cells may also be enriched using magnetic beads wherein the beads contain a ligand or antibody to a reconstituted cell surface protein or receptor.
  • the methods and compositions of the invention can be used to increase targeted insertion of a sequence of interest.
  • Cells can be modified with one or more of the desired zinc finger nuclease in the presence of the reporter and a donor sequence wherein following successful DNA cleavage by the nuclease, the donor sequence is incorporated either by homology-directed repair (HDR) or capture by end-joining.
  • HDR homology-directed repair
  • kits suitable for the identification, isolation and optimization of zinc finger nucleases as well as for targeted nucleic acid insertion or deletion into the genome of a cell.
  • nuclease comprises a zinc finger nuclease (ZFN).
  • ZFN zinc finger nuclease
  • ZFNs targeted to CCR5, GFP, WAS and Factor IX were designed and incorporated into plasmids vectors essentially as described in Urnov et al. (2005) Nature 435(7042):646-651 , Perez et al (2008) Nature Biotechnology 26(7): 808-816 , and United States Patent Publication No: 2008/0131962 or were obtained from Sigma Aldrich.
  • These ZFNs were constructed and tested by ELISA and the SurveyorTM (Transgenomics) Cel-1 assay ("Cel-1”) as described in Miller et al. (2007) Nat. Biotechnol. 25:778-785 and U.S. Patent Publication No. 20050064474 and International Patent Publication WO2005/014791 .
  • United States Provisional Application No. 61/212,265 relating to ZFNs targeted to Factor IX
  • United States Patent Publication No: 2008/0159996 relating to CCR5 -specific ZFNs.
  • a single-stranded annealing (SSA) reporter construct was assembled with two halves of the gfp gene separated by ZFN binding sequences in the middle ( Figure 1A ). Briefly, in this construct, 430 base pairs (bp) of the first half at the 3' end are identical to 430 bp of the 5' sequence of the second half. The first half has 146 bp unique sequences starting with the first Met codon ATG and the second half has 146 unique bp ending with the stop codon.
  • the ZFN binding sequence in the middle changes depending on the target sequence of ZFNs to be tested.
  • One or more ZFN binding sites can be inserted into the construct allowing one reporter construct to be used for screening more than one nuclease.
  • a construct may contain the target sequence for a control pair of ZFNs as well as the target for an unknown nuclease.
  • a CMV promoter lies in front of the gfp sequence and a polyA sequence follows the second half of the gfp sequence.
  • This plasmid also contains a Kanamycin resistance gene for propagation in bacteria.
  • a CCR5-specific ZFN binding site was inserted into the SSA reporter described above and GFP activity assayed in CHO-S cells following transfection by Amaxa nucleofection with the reporter construct and a pair of ZFNs targeting the inserted sequence. Reporter activity was measured as percentage of cells expressing GFP.
  • this assay showed a good dose response to both the amount of ZFN and the amount of SSA reporter ( Figure 1B and 1C ).
  • the GFP signal is most robust 48 to 72 hours after transfection ( Figure 1D ).
  • the optimal amount of ZFN and GFP-SSA reporter construct determined were used in subsequent experiments.
  • the correlation between ZFN activities on the SSA reporter with that at the endogenous target sequence in the genome was also determined.
  • a reporter as described in Example 2 was generated with multiple ZFN target sites of the human WAS gene (NCBI GeneID: 7454) and evaluated for GFP expression upon introduction of the appropriate ZFNs expression plasmids.
  • K562 cells were transfected with optimized amount of ZFN expression plasmid and a WAS GFP-SSA reporter construct. A third of the cells were taken 2 days after transfection and GFP signal was measured.
  • the rest of the cells were harvested 3 days after transfection and were used to analyze NHEJ activity at the endogenous WAS gene as a result of ZFN treatment, where NHEJ activity was assayed with the SurveyorTM nuclease as described, for example, in U.S. Patent Publication Nos. 20080015164 ; 20080131962 and 20080159996 (hereafter referred to as the "Cel-1 assay").
  • K562 cells were transfected with ZFN targeting the Factor IX gene and the appropriate SSA reporter construct (containing the Factor IX ZFN target sequence) and then were sorted by FACS 3 days after transfection.
  • ZFN and reporter construct transfected K562 cells were stained with propidium iodide (PI) for 5 minutes before FACS analysis. Gated FACS analysis showed that 0.3% the population expressed the highest levels of GFP, 0.9% of the population expressed mid level amounts of GFP and 2.3% of the cells expressed GFP at lower, but still detectable levels.
  • PI propidium iodide
  • NHEJ activity As shown in Figure 3 and Table 1 below, NHEJ activity, as determined via the Cel-1 assay, was increased in both FACS sorted (S) and unsorted (U) cells in the presence of the ZFN, as compared to cells transfected with the reporter plasmid only (G). Sorted cells showed higher NHEJ activity (see Figure 3 and Table 1 below).
  • Table 1 Sample NHEJ (%) GFP reporter alone (G) 0.26 GFP reporter + ZFN (FACS sorted) 40.79 GFP reporter + ZFN (unsorted by FACS) 17.90
  • the SSA reporter system was also tested in HeLa cells and PBMC cells with CCR5-specific ZFNs. Briefly, experiments were conducted as described above for K562 cells in HeLa cells. In addition, PBMC cells were transduced with adenoviruses expressing CCR5-specific ZFNs and a GFP-SSA reporter construct.
  • Single cell cloning of ZFN modified knock-out cells by standard limiting dilution can require the screening of hundreds, if not thousands, of clones.
  • Using conventional gene targeting strategies to knockout a gene without the aid of ZFNs may take several rounds of screening wherein the investigator must screen >100,000 cell clones. Therefore, we further tested if it is possible to efficiently isolate knockout cell clones by enriching for cells that had successfully reconstituted the reported gene by SSA.
  • clones were FACS analyzed for GFP expression 41 days after transfection. Results are shown in Table 3 below.
  • Table 3 FACS/SSA clone information GFP gating clone genotype GFP signal at day 41 Low P096 KO - Low P097 KO - Low P098 KO - Low P099 WT - Low P100 WT - Medium P101 KO - Medium P102 HET - Medium P103 KO - High P104 KO - High P105 KO - High P106 KO + High P107 KO -
  • the genotypes of 31 SSA/CCRS-specific ZFN-modified HeLa cell clones were also determined as set forth above. Of these clones, 13 exhibited wild type (WT) genotype, 11 were heterozygous (HET) for ZFN modifications and 7 were knockouts (KO).
  • Example 6 Enrichment and isolation of cells with ZFN-mediated targeted insertion
  • Endogenous targets have been modified by targeted insertion of an exogenous sequence (donor molecule) perhaps using homology directed repair (HDR) mediated by a ZFN.
  • HDR homology directed repair
  • the SSA assay was tested to determine if it could be used to enrich such targeted integration events as follows.
  • K562 cells were transfected using standard techniques with a small "patch" donor molecule in addition to CCR5-specific ZFNs and a GFP-SSA reporter construct.
  • the "patch" donor included 51 bp exogenous sequence between the two ZFN binding sites and was flanked by CCR5 gene sequence on both sides, which served as arms of homology for introducing the patch donor into the endogenous CCR5 locus.
  • the patch donor also included a novel BglII restriction site for PCR based restriction fragment length polymorphism (RFLP) analysis (see Urnov et. al. (2005) Nature 435:646-651 . Moehle et. al. (2006) PNAS 104:3055-3060 ; U.S.).
  • RFLP restriction fragment length polymorphism
  • NHEJ activity As shown in Figures 5A and 5B and Table 4 below, NHEJ activity, as determined by the Cel-1 assay, was increased in both FACS-sorted and unsorted cells ( Figure 5A ) in comparison with control reactions of either a mock transfection (no DNA) or a reporter construct only transfection (no ZFNs).
  • targeted integration of the patch donor was also increased in both sorted and unsorted cells as compared to controls ( Figure 5B ).
  • cells that had undergone targeted insertion of the patch sequence via HDR mediated targeted integration can be enriched by this method.
  • single cell clones with the desired targeted integration (TI) event were isolated as follows. 3 days after transfection, single cells were sorted into 96-well plates according to the gated gfp signal and allowed to grow. PCR based RFLP analysis described above was used to genotype the clones.
  • enrichment for ZFN-modified cells can be achieved by sorting cells for expression of a functional SSA reporter. Accordingly, enrichment capability of SSA sorting as compared to enrichment by GFP expression was compared as follows.
  • K562 cells were transfected with a ZFN expression plasmid along with a gfp expression plasmid to mimic the GFP expression level achieved using the ZFN-mediated reconstituted GFP-SSA reporter system.
  • Cells transfected with the ZFN expression plasmid, and then either with the GFP expression plasmid or with GFP-SSA reporter were sorted by FACS with identical settings 3 days after transfection.
  • the methods described herein do not involve drug selection, the derived single cell clones are not expected to have the SSA reporter gene integrated into their genomes.
  • the SSA assays as described herein was also used to screen a large set of ZFNs that were specific for several different target genes where the ability to induce NHEJ at the target was compared to GFP-SSA reporter activity.
  • the appropriate SSA reporter constructs for the ZFNs were generated as described above and tested as described above in K562 cells in 96-well plates. The number of ZFN pairs specific for particular target genes tested is listed in Table 7 as 'ZFN pairs'. ZFNs that gave a GFP signal yield higher than 50% of the CCR5-specific ZFNs signal were scored as positive. (see Table 7 below, "SSA+”) The ZFNs were scored NHEJ positive if they showed >1% NHEJ activity.
  • SSA based screening always correctly identified the positive hits as determined by NHEJ.
  • the ZFNs that scored high in the SSA assay also tended to have higher NHEJ activity at the endogenous locus.
  • Table 7 Target gene ZFN pairs SSA+ NHEJ+ %False+ False- NHEJ rank (SSA rank) A 16 9 7 22 0 1(1) B 19 12 11 8 0 1(2), 2(1) C 9 1 1 0 0 1(1) D 16 9 4 56 0 1(2), 2(1) E 8 6 5 17 0 1(5), 2(1), 3(2), 4(5) F 9 3 1 67 0 1(3), FP(1), FP(2) G 8 2 1 50 0 1(2), FP(1) H 9 7 5 29 0 1(1) Total 94 49 35 29 0 *FP - false positive
  • the SSA reporter system was also used to derive single cell clones in a high throughput fashion. Briefly, K562 cells were transfected with a panel of ZFNs targeting different genes and their corresponding SSA reporter constructs in 96-well format using Amaxa Shuttle. The NHEJ activity of the ZFNs were determined by the Cel-I assay 3 days after transfection. Cells were FACS sorted also 3 days after transfection into individual clones on 96-well plates. When the clones grew up, they were genotyped as described in Example 5 by PCR amplification of the target sequence followed by cloning and sequence analysis of the PCR product. Cell clones without any unmodified copy of the ZFN target sequence are designated KO clones. The frequency of KO clones of all clones analyzed are listed as the last column of Table 8.
  • Example 10 Enrichment of cells using a antibiotic resistance SSA reporter
  • a SSA reporter gene was constructed using the puromycin gene.
  • the puromycin SSA reporter was build similarly to the GFP SSA reporter described above.
  • the first 452 bp and last 422 bp of the puromycin resistance gene were interlinked with the ZFN targeting sites.
  • the ZFN used targets the CHO Bax gene (see, U.S. Patent Application No. 12/456,043 ).
  • HeLa cells were transfected by Amaxa nucleofection with plasmids as indicated in Figure 8 .
  • Cells were replated 24 hours after transfection in medium either with or without 1 ⁇ g/ml puromycin. Cell medium was replaced to regular medium 72 hours after transfection. Samples from different time point were collected and subjected to Cel-I assay analysis as described above.
  • Figure 8 shows a clear increase of NHEJ activity in SSA enriched sample 15 days after transfection, as measured by the Cel-I assay.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP10794489.4A 2009-06-30 2010-06-29 Rapid screening of biologically active nucleases and isolation of nuclease-modified cells Active EP2449135B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US26987109P 2009-06-30 2009-06-30
PCT/US2010/001858 WO2011002503A1 (en) 2009-06-30 2010-06-29 Rapid screening of biologically active nucleases and isolation of nuclease-modified cells

Publications (3)

Publication Number Publication Date
EP2449135A1 EP2449135A1 (en) 2012-05-09
EP2449135A4 EP2449135A4 (en) 2012-11-28
EP2449135B1 true EP2449135B1 (en) 2016-01-06

Family

ID=43411343

Family Applications (1)

Application Number Title Priority Date Filing Date
EP10794489.4A Active EP2449135B1 (en) 2009-06-30 2010-06-29 Rapid screening of biologically active nucleases and isolation of nuclease-modified cells

Country Status (7)

Country Link
US (4) US20110014616A1 (zh)
EP (1) EP2449135B1 (zh)
JP (1) JP5798116B2 (zh)
AU (1) AU2010266705B2 (zh)
CA (1) CA2765488C (zh)
HK (1) HK1170010A1 (zh)
WO (1) WO2011002503A1 (zh)

Families Citing this family (44)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2206723A1 (en) * 2009-01-12 2010-07-14 Bonas, Ulla Modular DNA-binding domains
US20110239315A1 (en) 2009-01-12 2011-09-29 Ulla Bonas Modular dna-binding domains and methods of use
CN103025344B (zh) 2010-05-17 2016-06-29 桑格摩生物科学股份有限公司 新型dna-结合蛋白及其用途
EP2392208B1 (en) * 2010-06-07 2016-05-04 Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Fusion proteins comprising a DNA-binding domain of a Tal effector protein and a non-specific cleavage domain of a restriction nuclease and their use
US20110201118A1 (en) * 2010-06-14 2011-08-18 Iowa State University Research Foundation, Inc. Nuclease activity of tal effector and foki fusion protein
US20120204282A1 (en) * 2011-02-04 2012-08-09 Sangamo Biosciences, Inc. Methods and compositions for treating occular disorders
KR20120096395A (ko) 2011-02-22 2012-08-30 주식회사 툴젠 뉴클레아제에 의해 유전자 변형된 세포를 농축시키는 방법
US10323236B2 (en) * 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
JP6144691B2 (ja) 2011-11-16 2017-06-07 サンガモ セラピューティクス， インコーポレイテッド 修飾されたｄｎａ結合タンパク質およびその使用
WO2013101877A2 (en) 2011-12-29 2013-07-04 Iowa State University Research Foundation, Inc. Genetically modified plants with resistance to xanthomonas and other bacterial plant pathogens
US20150044192A1 (en) * 2013-08-09 2015-02-12 President And Fellows Of Harvard College Methods for identifying a target site of a cas9 nuclease
WO2015023982A1 (en) 2013-08-16 2015-02-19 Massachusetts Institute Of Technology Selective delivery of material to cells
US9359599B2 (en) 2013-08-22 2016-06-07 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US9388430B2 (en) 2013-09-06 2016-07-12 President And Fellows Of Harvard College Cas9-recombinase fusion proteins and uses thereof
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
US9340799B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College MRNA-sensing switchable gRNAs
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
AU2014368982B2 (en) * 2013-12-19 2021-03-25 Amyris, Inc. Methods for genomic integration
EP3177718B1 (en) 2014-07-30 2022-03-16 President and Fellows of Harvard College Cas9 proteins including ligand-dependent inteins
CN107109362A (zh) 2014-10-31 2017-08-29 麻省理工学院 递送生物分子至免疫细胞
JP7278027B2 (ja) * 2015-01-12 2023-05-19 マサチューセッツ インスティテュート オブ テクノロジー マイクロ流体送達による遺伝子編集
WO2016132122A1 (en) * 2015-02-17 2016-08-25 University Of Edinburgh Assay construct
CA2988996A1 (en) 2015-07-09 2017-01-12 Massachusetts Institute Of Technology Delivery of materials to anucleate cells
US11613759B2 (en) 2015-09-04 2023-03-28 Sqz Biotechnologies Company Intracellular delivery of biomolecules to cells comprising a cell wall
EP3365356B1 (en) 2015-10-23 2023-06-28 President and Fellows of Harvard College Nucleobase editors and uses thereof
GB2568182A (en) 2016-08-03 2019-05-08 Harvard College Adenosine nucleobase editors and uses thereof
AU2017308889B2 (en) 2016-08-09 2023-11-09 President And Fellows Of Harvard College Programmable Cas9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
KR102622411B1 (ko) 2016-10-14 2024-01-10 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 핵염기 에디터의 aav 전달
WO2018119359A1 (en) 2016-12-23 2018-06-28 President And Fellows Of Harvard College Editing of ccr5 receptor gene to protect against hiv infection
US20200318166A1 (en) * 2017-01-18 2020-10-08 Altius Institute For Biomedical Sciences Multiplexed Screening
EP3583203B1 (en) 2017-02-15 2023-11-01 2seventy bio, Inc. Donor repair templates multiplex genome editing
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
WO2018165629A1 (en) 2017-03-10 2018-09-13 President And Fellows Of Harvard College Cytosine to guanine base editor
EP3601562A1 (en) 2017-03-23 2020-02-05 President and Fellows of Harvard College Nucleobase editors comprising nucleic acid programmable dna binding proteins
WO2018209320A1 (en) 2017-05-12 2018-11-15 President And Fellows Of Harvard College Aptazyme-embedded guide rnas for use with crispr-cas9 in genome editing and transcriptional activation
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
EP3676376A2 (en) 2017-08-30 2020-07-08 President and Fellows of Harvard College High efficiency base editors comprising gam
KR20200121782A (ko) 2017-10-16 2020-10-26 더 브로드 인스티튜트, 인코퍼레이티드 아데노신 염기 편집제의 용도
BR112021018606A2 (pt) 2019-03-19 2021-11-23 Harvard College Métodos e composições para editar sequências de nucleotídeos
DE112021002672T5 (de) 2020-05-08 2023-04-13 President And Fellows Of Harvard College Vefahren und zusammensetzungen zum gleichzeitigen editieren beider stränge einer doppelsträngigen nukleotid-zielsequenz
WO2023081756A1 (en) 2021-11-03 2023-05-11 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Precise genome editing using retrons
WO2023141602A2 (en) 2022-01-21 2023-07-27 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
WO2024044723A1 (en) 2022-08-25 2024-02-29 Renagade Therapeutics Management Inc. Engineered retrons and methods of use

Family Cites Families (55)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9011A (en) * 1852-06-15 Improvement
US5422251A (en) 1986-11-26 1995-06-06 Princeton University Triple-stranded nucleic acids
US5176996A (en) 1988-12-20 1993-01-05 Baylor College Of Medicine Method for making synthetic oligonucleotides which bind specifically to target sites on duplex DNA molecules, by forming a colinear triplex, the synthetic oligonucleotides and methods of use
US5420032A (en) * 1991-12-23 1995-05-30 Universitge Laval Homing endonuclease which originates from chlamydomonas eugametos and recognizes and cleaves a 15, 17 or 19 degenerate double stranded nucleotide sequence
US5356802A (en) 1992-04-03 1994-10-18 The Johns Hopkins University Functional domains in flavobacterium okeanokoites (FokI) restriction endonuclease
US5436150A (en) 1992-04-03 1995-07-25 The Johns Hopkins University Functional domains in flavobacterium okeanokoities (foki) restriction endonuclease
US5487994A (en) 1992-04-03 1996-01-30 The Johns Hopkins University Insertion and deletion mutants of FokI restriction endonuclease
US5792632A (en) * 1992-05-05 1998-08-11 Institut Pasteur Nucleotide sequence encoding the enzyme I-SceI and the uses thereof
JP4012243B2 (ja) 1994-01-18 2007-11-21 ザ スクリップス リサーチ インスティチュート 亜鉛フィンガータンパク質誘導体およびそのための方法
US6242568B1 (en) 1994-01-18 2001-06-05 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6140466A (en) 1994-01-18 2000-10-31 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
AU698152B2 (en) * 1994-08-20 1998-10-22 Gendaq Limited Improvements in or relating to binding proteins for recognition of DNA
GB9824544D0 (en) 1998-11-09 1999-01-06 Medical Res Council Screening system
US5789538A (en) 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
FI105922B (fi) * 1995-04-06 2000-10-31 Fortum Oil & Gas Oy Termoplastinen biohajoava polyesteri, menetelmä sen valmistamiseksi ja siitä valmistetut tuotteet
US5925523A (en) * 1996-08-23 1999-07-20 President & Fellows Of Harvard College Intraction trap assay, reagents and uses thereof
GB9703369D0 (en) 1997-02-18 1997-04-09 Lindqvist Bjorn H Process
GB2338237B (en) 1997-02-18 2001-02-28 Actinova Ltd In vitro peptide or protein expression library
GB9710807D0 (en) 1997-05-23 1997-07-23 Medical Res Council Nucleic acid binding proteins
GB9710809D0 (en) 1997-05-23 1997-07-23 Medical Res Council Nucleic acid binding proteins
US6534242B2 (en) 1997-11-06 2003-03-18 Canon Kabushiki Kaisha Multiple exposure device formation
US6410248B1 (en) 1998-01-30 2002-06-25 Massachusetts Institute Of Technology General strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites
CA2321938C (en) 1998-03-02 2009-11-24 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6140081A (en) * 1998-10-16 2000-10-31 The Scripps Research Institute Zinc finger binding domains for GNN
US7013219B2 (en) * 1999-01-12 2006-03-14 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6453242B1 (en) 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
US6794136B1 (en) 2000-11-20 2004-09-21 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
US6196012B1 (en) * 1999-03-26 2001-03-06 Carrier Corporation Generator power management
US20020061512A1 (en) 2000-02-18 2002-05-23 Kim Jin-Soo Zinc finger domains and methods of identifying same
WO2001088197A2 (en) 2000-05-16 2001-11-22 Massachusetts Institute Of Technology Methods and compositions for interaction trap assays
JP2002060786A (ja) 2000-08-23 2002-02-26 Kao Corp 硬質表面用殺菌防汚剤
GB0108491D0 (en) 2001-04-04 2001-05-23 Gendaq Ltd Engineering zinc fingers
WO2003016496A2 (en) 2001-08-20 2003-02-27 The Scripps Research Institute Zinc finger binding domains for cnn
CA2474486C (en) * 2002-01-23 2013-05-14 The University Of Utah Research Foundation Targeted chromosomal mutagenesis using zinc finger nucleases
WO2003078619A1 (en) * 2002-03-15 2003-09-25 Cellectis Hybrid and single chain meganucleases and use thereof
AU2003218382B2 (en) 2002-03-21 2007-12-13 Sangamo Therapeutics, Inc. Methods and compositions for using zinc finger endonucleases to enhance homologous recombination
AU2003298574B2 (en) 2002-09-05 2008-04-24 California Institute Of Technology Use of chimeric nucleases to stimulate gene targeting
EP2559759A1 (en) * 2003-01-28 2013-02-20 Cellectis Custom-made meganuclease and use thereof
US8409861B2 (en) 2003-08-08 2013-04-02 Sangamo Biosciences, Inc. Targeted deletion of cellular DNA sequences
ES2808687T3 (es) 2003-08-08 2021-03-01 Sangamo Therapeutics Inc Métodos y composiciones para escisión dirigida y recombinación
US7888121B2 (en) * 2003-08-08 2011-02-15 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US7972854B2 (en) 2004-02-05 2011-07-05 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US20060063231A1 (en) 2004-09-16 2006-03-23 Sangamo Biosciences, Inc. Compositions and methods for protein production
AU2006224248B2 (en) * 2005-03-15 2011-01-06 Cellectis I-Crei meganuclease variants with modified specificity, method of preparation and uses thereof
US20090068164A1 (en) * 2005-05-05 2009-03-12 The Ariz Bd Of Regents On Behald Of The Univ Of Az Sequence enabled reassembly (seer) - a novel method for visualizing specific dna sequences
JP2009502170A (ja) * 2005-07-26 2009-01-29 サンガモ バイオサイエンシズ インコーポレイテッド 外来核酸配列の標的化された組込み及び発現
DK2662442T3 (en) * 2005-10-18 2015-07-06 Prec Biosciences Rationally designed mechanuclease with altered dimer formation affinity
WO2007049095A1 (en) * 2005-10-25 2007-05-03 Cellectis Laglidadg homing endonuclease variants having mutations in two functional subdomains and use thereof
JP5266210B2 (ja) 2006-05-25 2013-08-21 サンガモ バイオサイエンシズ インコーポレイテッド 改変開裂ハーフドメイン
EP2447279B1 (en) * 2006-05-25 2014-04-09 Sangamo BioSciences, Inc. Methods and compositions for gene inactivation
EP2188384B1 (en) 2007-09-27 2015-07-15 Sangamo BioSciences, Inc. Rapid in vivo identification of biologically active nucleases
US8597912B2 (en) * 2008-06-10 2013-12-03 Sangamo Biosciences, Inc. Methods and compositions for generation of Bax-and Bak-deficient cell lines
EP2206723A1 (en) * 2009-01-12 2010-07-14 Bonas, Ulla Modular DNA-binding domains
PT2816112T (pt) * 2009-12-10 2018-11-20 Univ Iowa State Res Found Inc Modificação do adn modificada pelo efector tal

Also Published As

Publication number Publication date
EP2449135A1 (en) 2012-05-09
US20130171657A1 (en) 2013-07-04
US20120237926A1 (en) 2012-09-20
JP5798116B2 (ja) 2015-10-21
AU2010266705B2 (en) 2014-06-05
US9115409B2 (en) 2015-08-25
CA2765488A1 (en) 2011-01-06
WO2011002503A1 (en) 2011-01-06
AU2010266705A1 (en) 2012-01-12
EP2449135A4 (en) 2012-11-28
US20110244474A1 (en) 2011-10-06
JP2012531909A (ja) 2012-12-13
US20110014616A1 (en) 2011-01-20
US9121072B2 (en) 2015-09-01
HK1170010A1 (zh) 2013-02-15
CA2765488C (en) 2018-01-02

Similar Documents

Publication Publication Date Title
EP2449135B1 (en) Rapid screening of biologically active nucleases and isolation of nuclease-modified cells
US9833479B2 (en) Gene correction of SCID-related genes in hematopoietic stem and progenitor cells
EP2188384B1 (en) Rapid in vivo identification of biologically active nucleases
US8772009B2 (en) Methods and compositions for increasing nuclease activity
US20160145645A1 (en) Targeted integration
US9809839B2 (en) Method for concentrating cells that are genetically altered by nucleases
US20160251411A1 (en) Host cell protein modification
KR20210141536A (ko) 타우 응집과 관련된 유전적 취약성을 식별하기 위한 CRISPR/Cas 드랍아웃 스크리닝 플랫폼
US20120329067A1 (en) Methods of Generating Zinc Finger Nucleases Having Altered Activity
WO2019089623A1 (en) Fusion proteins for use in improving gene correction via homologous recombination
JP2004518419A (ja) 基質連結型指向性進化（ＳＬｉＤＥ）
US20180238877A1 (en) Isolation of antigen specific b-cells

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20111216

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20121026

RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 15/90 20060101ALI20121022BHEP

Ipc: C12Q 1/68 20060101AFI20121022BHEP

REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1170010

Country of ref document: HK

17Q First examination report despatched

Effective date: 20131028

REG Reference to a national code

Ref country code: DE

Ref legal event code: R079

Ref document number: 602010030015

Country of ref document: DE

Free format text: PREVIOUS MAIN CLASS: C12Q0001680000

Ipc: C12N0015630000

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 5/10 20060101ALI20150724BHEP

Ipc: C12N 15/63 20060101AFI20150724BHEP

INTG Intention to grant announced

Effective date: 20150826

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 768920

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160215

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602010030015

Country of ref document: DE

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20160106

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 768920

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160106

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 7

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

REG Reference to a national code

Ref country code: HK

Ref legal event code: GR

Ref document number: 1170010

Country of ref document: HK

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160407

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160506

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160506

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602010030015

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

26N No opposition filed

Effective date: 20161007

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160406

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160630

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160630

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 8

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160629

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 9

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20100629

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160629

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

Ref country code: MT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160106

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20230626

Year of fee payment: 14

Ref country code: DE

Payment date: 20230626

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20230620

Year of fee payment: 14

Ref country code: GB

Payment date: 20230627

Year of fee payment: 14